Figure 5. N-acetyl cysteine prevents loss of MAP2⁺ neuronal profiles after dual MG132/MG132 hits.

Primary hippocampal cultures were treated on DIV5 with the 1^st hit of MG132 and on DIV6 with the 2^nd hit of MG132 in the absence or presence of N-acetyl cysteine (NAC). Viability was assayed 48h later on DIV8 by (A) the nuclear stain DRAQ5, (B) the In-Cell Western assay for MAP2 levels, and (C) the Cell Titer Glo luminescent assay for ATP. Representative DRAQ5 and MAP2 images are shown in panels I and J. Panels A–C are shown as scatterplots in Supplemental Figure 3 (G–I) to show all individual data points. (D) The same treatments as shown in panels A–C were repeated and glutathione (GSH) levels were measured by the In-Cell Western technique and expressed as a function of the nuclear DRAQ5 stain. A representative image of the glutathione In-Cell Western is shown in panel K. (E–H) Data in panels A–D are expressed as a function of the 0 μM 2^nd hit group in order to statistically evaluate the effect of dual hits. NAC mitigated MG132 toxicity in response to single hits of MG132. The toxicity of dual MG132 hits was not ameliorated by NAC according to the DRAQ5 and ATP assays, but was mitigated by NAC in the MAP2 assay, which is specific for neuronal elements. The transformed data in panel H reveal that NAC prevents synergistic loss of GSH in response to dual MG132/MG132 hits. Shown are the mean and SEM of 3–5 independent experiments, each performed in triplicate. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 versus 0 μM 1^st hit; +p ≤ 0.05, ++p ≤ 0.01, +++p ≤ 0.001 versus 0 μM 2^nd hit; ~p ≤ 0.05, ~~p ≤ 0.01, ~~~p ≤ 0.001 versus 0 μM NAC; three-way ANOVA followed by Bonferroni post hoc correction.